Abstract: The transdifferentiation of human fibroblasts to induced neurons and other cell types has significant impact on many practical applications and fundamental understanding of cell biology. It suggests that cell type identity is largely determined and maintained by the cell's transcription regulatory network, which can be readily rewired by a few key transcription factors to direct the same genome to express a different cell type. The low efficiency of these relatively fast conversions, which generally manifest themselves within days, suggests that conditions additional to the requisite transcription factors must be met to enable highly efficient cellular reprogramming. Identification of these kinetic barriers would reveal significant mechanistic insights into cell lineage reprogramming and produce highly efficient ways to generate many different types of useful cells from readily available cells such as fibroblasts. Increasing evidence indicates that there are significant differences between human and animals in many aspects of biomedical research. It is thus very important to develop human cell models to study various human diseases that are highly relevant to the missions of the Department of Veterans Affairs. To that end, we have significantly improved the technique to reprogram human fibroblasts to induced dopaminergic neurons by defined factors (Ascl1, Nurr1, Lmx1a and miR-124). We found that cell cycle arrest at G1 phase and p53 knockdown in conjunction with the appropriate extracellular environment dramatically increased the efficiency of converting human primary fibroblasts to induced dopaminergic neurons. In this proposal, we will study the molecular mechanisms by which cell cycle arrest, p53 knockdown and extracellular environment affect the reprogramming process. Knowledge gained from the study will help us to understand the fundamental plasticity of cell lineage determination and will provide mechanistic insights into the direct transdifferentiation of human fibroblasts to cells that are useful for many areas of biomedical research important for the mission of the Department of Veterans Affairs.